Compositions and methods for in vitro diagnostic tests including sulfonic acid

ABSTRACT

The invention provides compositions, kits, and methods for performing colorimetric analysis. A substrate is reacted to generate a chromogenic reaction product, and a reaction stop reagent that is a sulfonic acid is added to stop and stabilize the reaction product. The absorbance properties of the chromogenic reaction product can be maintained over significantly longer periods of time of that of conventional reagents and methods. The sulfonic acid can be used in assays such as ELISAs in order to provide a more accurate and safer detection of analytes in a biological sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present non-provisional application is a continuation of U.S. patentapplication Ser. No. 13/849,056, filed on Mar. 22, 2013, entitledCOMPOSITIONS AND METHODS FOR IN VITRO DIAGNOSTIC TESTS INCLUDINGSULFONIC ACID COMPOUND, which claims the benefit of U.S. ProvisionalApplication No. 61/614,602, filed on Mar. 23, 2012, entitledCOMPOSITIONS AND METHODS FOR IN VITRO DIAGNOSTIC TESTS INCLUDINGSULFONIC ACID COMPOUND, which applications are incorporated herein byreference in its entirety.

FIELD

The invention relates to compositions and methods for in vitrodiagnostic tests and in vitro colorimetric tests.

BACKGROUND

Research and diagnostic procedures require rapid, accurate andqualitative and/or quantitative determinations of substances(“analytes”) that are present in biological samples, such as biologicaltissues or fluids, at low concentrations. For example, the presence ofdrugs, narcotics, hormones, steroids, polypeptides, prostaglandins orinfectious organisms in blood, urine, saliva, dental plaque, gingivalcrevicular fluid, and other biological specimens is desirably determinedin an accurate and rapid fashion for suitable diagnosis or treatment.

In many cases, an analyte is identified in a sample using a compoundthat specifically recognizes the chemical features of the analyte.Often, monoclonal antibodies specific for one or more chemical epitopeson an analyte are used. The complex formed between the antibody andanalyte can be detected by a variety of known methods. The most commonlyused methods employ a signal generating moiety of some type which iseither already attached to the antibody, or becomes attached to theantibody through further reaction. For example, in the formation of acomplex of biotin with avidin, the complex may be detected using a labelon either the avidin or biotin molecule. Such a label can be aradioisotope or an enzyme conjugated with the avidin or biotin.Alternatively, the avidin-biotin complex might be detected by furtherreaction with a labeled molecule which is specific to either or bothparts of the complex. It is commonly known to do the same with antigensand their corresponding antibodies.

In diagnostic tests designed to be rapid and easy to use with moderatetraining in a doctor's office or clinic, the specific binding ligand ofinterest (such as an antigen from an infectious agent) is often detectedusing colorimetric, fluorescent or chemiluminescent signals resultingfrom reaction of the enzyme label with its corresponding substrate.

There is a need to produce the signal quickly and intensely if theligand is present. This is commonly done using a colorimetric detectionreagent. Upon addition of the detection reagent a colored product isproduced. In many types of assay, the generation of color is notlimited. In order to optimally quantitate the result, a stop reagent isemployed to stop the formation of color and hold it at a stable level toallow for accurate quantitation. Acids such as sulfuric acid andhydrochloric acid can be used to stop the production of detectablesignal when peroxidase is used as a label in specific binding reactions.However, the use of these acids has problems associated with it, namelycorrosivity, short signal duration, and toxicity.

The investigator has discovered there is a need to provide anon-corrosive stop reagent and a stop reagent that allows for anextended dynamic range and a longer, more stable signal time thanprevious acids have provided.

SUMMARY

Generally, the invention provides compositions, kits, and methods fordetermination of an analyte in a sample which uses a chromogenicsubstrate detectable by colorimetric analysis, and a stop reagent forstopping and stabilizing a reaction composition comprising a chromogenicreaction product. The stop reagent is a sulfonic acid.

Experimental studies associated with the invention have unexpectedlyfound that the sulfonic acid stop reagents stabilize absorbanceproperties of the chromogenic reaction product over significantly longerperiods of times as compared to assays using conventional stop reagents.In addition, the stop reagents of the invention allowed for colorimetricanalysis, especially at high analyte levels, to be performed over anextended dynamic range. Advantageously, the stop reagents of theinvention do not have the corrosivity characteristics of sulfuric andhydrochloric acids, which make the components and compositions of thekits and methods of the invention more amenable to handling.

Accordingly, one aspect of the invention provides a kit with componentsfor performing a colorimetric assay. The kit comprises a chromogenicsubstrate capable of forming a chromogenic reaction product detectableusing colorimetric analysis, and a compound that stops and stabilizesthe chromogenic reaction product, the compound being a sulfonic acid.The kit can optionally include other components like one or more redoxcompounds, such as peroxidases, peroxidase substrates, oxidases andoxidase substrates, which can be used to convert the chromogenicsubstrate into the chromogenic reaction product. Another optionalcomponent is an analyte binding member, such as an antibody, capable ofspecific recognition of an analyte in a biological sample. Otheroptional components in the kit include vessels, such as multiwellplates, in which the colorimetric analysis can be carried out and thechromogenic reaction product read using spectrophotometric equipment.

The invention also provides a composition comprising the sulfonic acidand chromogenic reaction product detectable using colorimetric analysis.The chromogenic reaction product can be stabilized in the presence ofthe sulfonic acid. The composition can optionally include othercomponents that can be used to generate the chromogenic reaction productsuch as a peroxidase, peroxidase substrate, oxidase, and oxidasesubstrate.

The method further provides methods for performing a colorimetricanalysis. The method includes, in the least, a step of adding thesulfonic acid to a composition containing the chromogenic reactionproduct. The addition of the sulfonic acid stabilizes the chromogenicreaction product which can then be colorimetrically analyzed usingequipment such as a spectrophotometer, or alternatively byvisualization. The method can optionally include one or more stepsupstream of the step of adding the compound with the sulfonic acidgroup. For example, the method can optionally include step(s) ofproviding a biological sample having an analyte, contacting the analytewith an analyte-binding compound, recognizing theanalyte/analyte-binding compound complex with one or more components,such as a peroxidase, peroxidase substrate, oxidase, and oxidasesubstrate that promote conversion of a substrate, and formation of thereaction product detectable using colorimetric analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the change in absorbance oftetramethylbenzidine (TMB) over time after the addition of variousacidic stop reagents to a color-generating reaction mixture of TMB,horseradish peroxidase (300 pg/well), and hydrogen peroxide.

FIG. 2 is a graph showing the change in absorbance oftetramethylbenzidine (TMB) over time after the addition of variousacidic stop reagents to a color-generating reaction mixture of TMB,horseradish peroxidase (40 pg/well), and hydrogen peroxide.

FIG. 3 is a graph showing the differences in absorbance oftetramethylbenzidine (TMB) at increasing amounts of analyte (asreflected by IgG concentration) measured at different times after theaddition of 0.5 M sulfuric acid stop reagent to a color-generatingreaction mixture of TMB, horseradish peroxidase, and hydrogen peroxide.

FIG. 4 is a graph showing the differences in absorbance oftetramethylbenzidine (TMB) at increasing amounts of analyte (asreflected by IgG concentration) measured at different times after theaddition of 0.15 M methanesulfonic acid stop reagent to acolor-generating reaction mixture of TMB, horseradish peroxidase, andhydrogen peroxide.

FIG. 5 is a graph showing the differences in absorbance oftetramethylbenzidine (TMB) at increasing amounts of analyte (asreflected by IgG concentration) measured at different times after theaddition of 0.2 M sulfamic acid stop reagent to a color-generatingreaction mixture of TMB, horseradish peroxidase, and hydrogen peroxide.

DETAILED DESCRIPTION

The embodiments of the present invention described herein are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather, the embodimentsare chosen and described so that others skilled in the art canappreciate and understand the principles and practices of the presentinvention.

All publications and patents mentioned herein are hereby incorporated byreference. The publications and patents disclosed herein are providedsolely for their disclosure. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate anypublication and/or patent, including any publication and/or patent citedherein.

The present invention is directed to compositions, kits, and methods forperforming a colorimetric assay using sulfonic acid. Sulfonic acid is acompound that includes one or more sulfonic acid group(s) (—SO₃H).Sulfuric acid (H₂SO₄) is not a sulfonic acid. In experiments associatedwith the invention, it has been found that use of the sulfonic acid stopreagent provides distinct benefits in assays in which a chromogenicreaction product is detected by colorimetric analysis. The sulfonic acidstop reagent of the invention have been shown to stabilize thechromogenic reaction product so the loss of signal intensity orabsorbance of the reaction product after the stop reagent is added isminimized. For example, after a color- or signal-generating reaction iscarried out, the stop reagent is added to the reaction composition tomaintain favorable color characteristics (e.g., as measured by opticalabsorbance) or signal intensity over extended periods of time. Bycomparison, more rapid changes in color characteristics (e.g., asmeasured by optical absorbance) are observed when known stop reagentssuch as sulfuric acid (as the only stop reagent in the composition) areused.

Minimizing loss of signal intensity or absorbance can provide a moreaccurate assessment of the amount of analyte in the sample. It can alsoafford the user with greater flexibility for carrying out colorimetricanalysis, and therefore the user can optionally make the analysisprocess more complex, can optionally increase the throughput of theanalysis (e.g., analyze more samples), or both. The decrease in signalintensity using known stop reagents such as sulfuric acid isparticularly dramatic when the sample includes a high amount of analyte.In further experimental studies associated with the invention, it wasshown that use of the sulfonic acid stop reagents of the inventiongreatly minimize the loss of signal intensity at high analyte levels.Accordingly, this improves the dynamic range of detection when stopreagents of the invention are used in a colorimetric analysis.

Exemplary sulfonic acid stop reagents comprise one or more sulfonic acidgroups (—SO₃H), but do not include sulfuric acid (H₂SO₄). Compounds thatcan be used as sulfonic acid stop reagents, including those describedherein, are commercially available or can be prepared according tomethods known in the art of chemical synthesis. Exemplary sulfonic acidstop reagents can include non-polymeric and polymeric compounds.

In some embodiments, the kits, composition, and methods use a sulfonicacid stop reagent of Formula I: R—SO₃H, wherein R is selected from thegroup consisting of alkyl, aminoalkylene, aminoalkyl, hydroxyalkyl,aryl, amine, fluoroalkyl, perfluoroalkyl, and

wherein R¹ is a branched or straight chain alkylene group.

Exemplary alkylsulfonic acids include those where R is methyl, ethyl, orpropyl, which provides methanesulfonic acid (methylsulfonic acid;CH₃SO₃H), ethanesulfonic acid (ethylsulfonic acid; CH₃CH₂SO₃H), andpropanesulfonic acid (propylsulfonic acid; CH₃CH₂CH₂SO₃H), respectively.

In some cases the sulfonic acid includes an amine group. For example, ifR is —NH₂ then the compound is sulfamic acid (also known asamidosulfonic acid, amidosulfuric acid, aminosulfonic acid, andsulfamidic acid; H₂NSO₃H). Other amine-group-containing sulfonic acidsare aminoalkylenesulfonic acids, such as aminomethanesulfonic acid,2-aminoethanesulfonic acid (taurine), and aminoproanesulfonic acid.

In some cases the sulfonic acid is an arylsulfonic acid, such asp-toluenesulfonic acid (PTSA; tosylic acid), xylenesulfonic acid, andcumenesulfonic acid.

In more specific embodiments, when R is

compounds of Formula II are provided:

with exemplary R¹ groups including methylene (—(CH₂)—), ethylene(—(CH₂CH₂)—), methylmethylene

dimethylmethylene

methylethylene

and dimethylethylene

where Formula II includes compounds such as 2-acrylamido-2-methylpropanesulfonic acid, 2-acrylamido propane sulfonic acid, and 2-acrylamidoethane sulfonic acid.

The invention also contemplates stop reagents having two or moresulfonic acid groups, such as disulfonic acids. Commercially availabledisulphonic acids include, for example, 2-naphthol-3,6-disulfonic acid,phenol disulfonic acid, ethanedisulfonic acid, and aniline disulfonicacid. Polymers containing two or more sulfonic acid groups, exemplifiedby those derived from acrylamide described, above, are alsocontemplated.

Optionally, the stop reagent of the invention can be referred to as a“sulfonic acid derivative” which refers to compounds represented bysulfonic acid compounds and formulas such as those described hereinwhich is not sulfuric acid.

In some embodiments, structures of exemplary sulfonic acids arerepresented in Table 1 below.

TABLE 1 Designation Structure dodecylbenzenesulfonic acid (DBSA)

4,5-dihydroxy-1,3-benzenedisulfonic acid

3-hydroxypropane-1-sulfonic acid

4,4′-diamino-2,2′-stilbenedisulfonic acid

methanesulfonic acid

ethanesulfonic acid

propanesulfonic acid

(4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) (HEPES)

3-(N-morpholino)propanesulfonic acid (MOPS)

3-(N-morpholino)-2-hydroxy-1- propanesulfonic acid (MOPSO)

perfluorooctanesulfonic acid

triflic acid

p-toluenesulfonic acid (PTSA)

sulfamic acid

taurine

The sulfonic acid can also be described in terms of its aciddissociation constant (pK_(a); or acid ionization constant). Sulfonicacid stop compounds of the invention may have more than one aciddissociation constant depending on the protonic or zwitterionic natureof the compound. For purposes of discussion, if the compound has morethan one acidic dissociation constant, the compound's pK_(a) refers toits lowest (most acidic) acid dissociation constant. In some cases thesulfonic acid stop compound has a pK_(a) in the range of about 4 to −3,or in the range of about 4 to about −2.5, or in the range of about 4 toabout −2, or in the range of about 3 to about −2.5, or in the range ofabout 2 to about −2.5, or even in the range of about 1 to about −2.5.

The sulfonic acid stop compound can also be described in terms of itsliquid solubility. Since colorimetric assays are commonly performed in awater-based solution, such as a buffered aqueous solution, in manyembodiments the sulfonic acid stop compound is at least slightly solublein water. Descriptive terms for solubility are standard terms used inthe art (see, for example, Remington: The Science and Practice ofPharmacy, 20^(th) ed. (2000), Lippincott Williams & Wilkins, BaltimoreMd.), with “slightly soluble” referring to compounds having a solubilityof 1 part agent per from 100 to 1000 parts of solvent; “sparinglysoluble” referring to compounds having a solubility of 1 part agent from30 to 100 parts of solvent; “soluble” referring to compounds having asolubility of at least 1 part compound per from 10 to 30 parts solvent,“freely soluble” refers to compounds having a solubility of at least 1part agent per from 1 to 10 parts solvent, and “very soluble” refers tohaving a solubility of greater than 1 part agent per from 1 partsolvent.

Compositions of the invention based on aqueous solutions can optionallybe formulated to include water soluble co-solvents or components thatcan improve the solubility of components in the composition (e.g., thestop reagent) if desired. Examples of useful optional co-solvents arealcohols (i.e. methanol, ethanol, propanol), polyalcohols (i.e.glycerol, propylene glycol), dimethylsulfoxide, dimethylformamide,acetonitrile, and similar solvents. Other solvents to improve solubilityare well known in formulation art for in vitro diagnostic applications,can optionally be included in compositions of the invention. Othersolubility-enhancing components such as described in U.S. ProvisionalNo. 61/751,652 (Jan. 11, 2013; Lundquist) can be included in thecomposition and/or kit of the invention.

In some cases, a sulfonic acid stop reagent of the invention can bemixed with one or more other sulfonic acid stop reagent(s) of theinvention. For example, kits, compositions, and methods of the inventioncan include a mixture of methanesulfonic acid and sulfamic acid.Optionally, a stop reagent of the invention can be mixed with one ormore other stop reagent(s) already known in the art of colorimetricassays.

Sulfonic acid stop reagents of the invention can be provided in solid orliquid form, depending on the properties of the compound, and thedesired arrangement of the kit or composition. If the sulfonic acid stopreagent is a liquid at ambient conditions, it can be supplied and/orused in neat form, or can be diluted with a suitable solvent, such aswater. In some embodiments the sulfonic acid stop reagent can besupplied in a dry form (e.g., powder, granule, pellet, etc.) and then bedissolved prior to use by using an appropriate solvent. In yet otherembodiments the sulfonic acid stop reagent can be dissolved by mixingwith a known liquid stop reagent (e.g. H₂SO₄).

The kits, compositions, and methods of the invention can also include achromogenic substrate (or a reaction product of the substrate;“chromogenic reaction product”) for colorimetric analysis. In an assay,the chromogenic substrate is subjected to a chemical reaction, such asone associated with an enzymatic reaction, resulting in a chromogenic“reaction product” detectable using colorimetric analysis. For example,in an assay, a chromogenic substrate can undergo chemical oxidation orreduction, or chemical modification by the addition, subtraction, orrearrangement of chemical groups on the chromogenic substrate. Thereaction product can be one where, for example, the oxidized or reducedform the chromogenic substrate, or a chemically-modified form of thechromogenic substrate, is detectable by colorimetric analysis.

In some embodiments the chromogenic substrate can be a compound that isuncolored, or one that has a first color, or one that does not emit asignal, or combinations thereof. The chromogenic reaction product can becolored, have a (second) color that is different than the first color,or emit a signal, or combinations thereof. In some cases the chromogenicreaction product is formed by the reaction of two starting compoundswhich produces a chromogenic substrate that is oxidized to produce acolored chromogenic reaction product.

A variety of known chromogenic substrates for colorimetric analysis canbe used in the kits, compositions, and methods of the invention.Chromogenic substrates contemplated for use with the current inventioninclude benzidine, ortho-tolidine, ortho-dianisidine, which can changecolor in the presence of peroxidase-containing components. In someembodiments, the kits, compositions, and methods of the invention use abenzidine derivative such as 3,3′,5,5′-tetramethylbenzidine. Benzidinederivatives include those of formula III:

where X, X′, Y, Y′, R, and R′ are independently hydrogen, alkyl, alkoxy,or combinations thereof, wherein the alkyl or alkoxy group(s), ifpresent, having up to six carbon atoms. In more specifically aspects offormula III, X, X′, Y, Y′, R, and R′ are independently hydrogen, alkyl,alkoxy, or combinations thereof, wherein the alkyl or alkoxy group(s),if present, containing four or less carbon atoms. Benzidine-typecompounds include 3,3′,5,5′-tetramethylbenzidine, o-tolidine,o-dianisidine, N,N,N′,N′-tetramethylbenzidine. See, for example, U.S.Pat. No. 6,376,252.

In the presence of horseradish peroxidase (HRP) and hydrogen peroxidetetramethylbenzidine (3,3′,5,5′-tetramethylbenzidine; TMB) is oxidizedto form colored products. As described by Josephy et al. (1982; J. Biol.Chem. 257:3669-3675), TMB (λ_(max) 285) is oxidized to form oxidizedTMB, a blue product (λ_(max) 652) which is a one-electron oxidationproduct, and then is further oxidized to form a yellow product (λ_(max)450), which is the two-electron oxidation product. Colorimetric analysisand measurement of the reaction product is typically carried out at 450nm, but can also be observed with the unaided human eye (ReactionSequence A).

Other chromogenic material includes Trinder reagents which use aphenylpyrazone derivative and a phenolic or an aromatic amine compound.In the chemistry of peroxidase reactions with Trinder reagents, twocolorless organic molecules form a colored product in the presence ofperoxidase and hydrogen peroxide. Exemplary phenylpyrazone derivativesinclude 4-aminoantipyrine, and exemplary phenols include cresol andsalicylamide; other exemplary phenylpyrazone derivatives and phenols aredescribed in U.S. Pat. No. RE29,498. Exemplary aromatic amines includeN,N-dialkylaniline compounds, N,N-dialkyl-m-toluidine compounds,toluidine and aniline sulphopropyl derivatives,N-ethyl-N-(3-sulphopropyl)-m-anisidine andN-ethyl-N-(2-hydroxyethyl)-m-toluidine, as described in U.S. Pat. No.5,206,006.

Other chromogenic material includes OPD (o-phenylenediamine) which is awater-soluble substrate for horseradish peroxidase (HRP) that produces ayellow-product detectable at 450 nm. Addition of acid to the OPDreaction product produces a yellow-orange with an absorbance maximum of492.

Tyramine is used as a substrate in dopamine-beta-hydroxylase (DBH)assays. DBH converts 3,4-dihydroxyphenylethylamine (dopamine) into theneurotransmitter norepinephrine. Tyramine is converted by DBH tooctopamine, which is then oxidized to parahydroxybenzaldehyde by sodiumperiodate. The oxidation is stopped by sodium metabisulfite.Parahydroxybenzaldehyde is then quantified by measuring its absorbanceat 330 nm.

The water-soluble HRP substrate2,2′-azino-di(3-ethylbenzthiazoline-6-sulfonate) (ABTS) yields a greenend product upon reaction with peroxidase. The green product has twomajor absorbance peaks, 410 nm and 650 nm. ABTS is less sensitive thanOPD and TMB in ELISA applications. It is less readily oxidized, and itscolor development is slower (approximately 20-60 minutes).

Other chromogenic material that can be used in the methods, kits, andcompositions of the invention includes the tetrazolium dye, MTT, whichcan be reduced to insoluble formazan, which is purple.

Aspects of some embodiments of the present disclosure relate to the useof the sulfonic acid stop reagent comprising with colorimetric assays.Generally, colorimetric analysis can be performed to determine thepresence and/or amount of an analyte in a sample. The term “analyte”refers to any substance or chemical constituent of a sample that isbeing analyzed. The analyte can be a natural compound, such as one thatis produced by an organism, or can be a non-natural compound, such as asynthetic compound used as a bio-affecting agent like a drug, apesticide, or herbicide. Methods, compositions, and kits of theinvention can be used for the determination of analytes in numerousindustrial applications including, but not limited to health care, foodmanufacture and processing, chemical analysis and production,agriculture, environmental control, and the like. In some aspects, anELISA (enzyme-linked immunosorbent assay) is used for detection of ananalyte, and the ELISA and/or or kit used to perform an ELISAincorporates the sulfonic acid stop reagent of the invention.

As described herein, in some modes of practice, a colorimetric analysisor colorimetric assay can be conducted using specialized equipment knownin the art to quantify and measure the wavelength and/or absorbance ofthe solution being analyzed. Such equipment includes, but is not limitedto UV-visible spectrophotometers and multiwell plate readers In someembodiments, the colorimetric analysis or colorimetric assay may beconducted, and endpoint determined, using the unaided human eye.

Various types of analytes can be detected and quantified in a sampleusing the methods of the invention. In some aspects, detection of theanalyte is facilitated by using an analyte binding member, such as anantibody. In other aspects, methods of the invention provide fordetection of an analyte without utilizing an analyte binding member.Non-limiting exemplary analytes include drugs, drug metabolites,biomarkers, hormones, antibiotics, food supplements, food additives,naturally occurring contaminants, dyes, microorganisms and their toxins,fungi, viruses, pesticides, herbicides, organic components of wastedischarges, tissue specific markers, tissue specific enzymes, cytokines,chemokines, growth factors, receptor ligands, enzymes, nucleic acids,lipids, and small organic molecules, such as glucose and peroxides.

For example, in the food processing and manufacturing industries forhumans, as well as domesticated and farm animals, ELISAs are used forthe detection of various analytes that are polypeptides, such as soyproteins and gluten, which can be allergens, and other compounds such asantibiotics and hormones.

As other non-limiting examples, in the health care industry ELISAs areused for the detection of various analytes in blood, urine, and otherbody fluids for the detection of analytes such as erythropoietin (EPO),adrenocorticotropic hormone (ACTH), calcitonin, parathyroid hormone(PTH), thyroid stimulating hormone (TSH), prostate-specific antigen(PSA), human chorionic gonadotropin (HCG), follicle stimulating hormone(FSH), growth hormone (GH).

In some embodiments, the kit, composition, or method of the invention isconfigured for detection of a nucleic acid analyte. Any type of nucleicacid capable of interacting with a complimentary nucleic acid sequencecan be detected as the analyte. Exemplary analytes include nucleic acidfragments (e.g., restriction digested DNA) of bacterial, viral, andeukaryotic DNA, including genomic DNA, plastid DNA, mitochondrial DNA,etc.; as well as mRNA (messenger RNA), iRNA (immune ribonucleic acid),ribozymes, siRNA (small interfering RNA), miRNA (micro RNA), and shRNA(short hairpin RNA).

The sample including an analyte to be detected can be a biological or anon-biological sample.

A biological sample can be any material taken from an organism such asbody fluid from a mammal, material derived from an organism, or a samplethat has organisms in it. Biological samples include certain tissues, orbody fluid such as blood, sputum, urine, saliva, mucus, vitreal fluid,synovial fluid, semen, cerebrospinal fluid, bone marrow, amniotic fluid,bile, sweat, etc. Biological samples can be obtained from patients andanalyzed for the absence or presence of analytes associated with diseasestates. Quantitation of an analyte can be used to determine the absence,presence or degree of a disease state. Biological samples can alsoinclude sections of tissues such as frozen sections taken forhistological purposes which can also be analyzed for analytes associatedwith disease states.

Other samples, including biological samples, can be those derived fromfermentation, cell culturing, bio-fuel production, wastewater treatment,and agriculture. These include food products such as milk, wine, beer,and the like; chemical streams, or waste streams from chemical plants,rivers, and the like. If the sample is initially complex, solid, orviscous, it can optionally be treated, such as by extraction, or it cambe dissolved or diluted in order to obtain a sample having theappropriate characteristics for use in the immunoassay.

An example of a non-biological sample is a composition of achemically-synthesized component, such as a synthetic drug for humantreatment or a synthetic pesticide for agricultural use.

Analyte detection and the chromogenic assay can be performed in asuitable assay vessel. An assay vessel is any suitable receptacle inwhich analyte detection, such as by ELISA, and the chromogenic assay canbe performed. The assay vessel can be made from material such as glass(e.g., surface modified glass), quartz, or plastic, such as polystyrene,polypropylene, and polycarbonate. Exemplary assay vessels are single andmulti-well plates, such as medium and smaller-welled plastic plates suchas 6, 24, 96, 384, and 1536 well plates. These are commonly known in theart as microtiter plates, microplates, or microwell plates. Exemplaryplates for use in chromogenic assays in each well can hold frommicroliter to milliliter volumes of liquid. Other types of assay vesselsthat can be used for analysis include capillary tubes. The assay vesselcan optionally be included in a kit, or can be supplied by the user tocarry out chromogenic assay methods as described herein.

For example, in some modes of practice, 96-well plates are used foranalyte detection and the chromogenic assay. A single well in a 96-wellplate generally holds up to about 300 μL to 350 μL of liquid, and all ora fraction of this volume can be used during steps in methods of theinvention. This can provide a convenient volume for steps in ELISAchromogenic assays, involving antibody, analyte, and enzymeimmobilizations, dispensing a biological sample, washing steps,dispensing a chromogenic substrate, and the addition of a stop reagentfor the colorimetric analysis. The area on the bottom of a 360 μL wellis about 0.32 cm².

In some cases, it is not necessary to identify the analyte (to bedetected) in the biological sample with an analyte binding member, suchas an antibody. Some assays use enzymes to measure certain biologicalsubstances in samples. For example, some assays use a peroxidase enzymeto measure peroxides present in a sample. Conversely, some assays detectthe presence of biological enzymes by using the enzyme's correspondingsubstrate. For example, addition of hydrogen peroxide to a sample can beused to test for peroxidase enzymatic activity in the sample.

In some aspects of the invention, the sulfonic acid stop reagent is usedin an enzyme-linked immunosorbent assay (ELISA). Generally, an ELISAuses a solid-phase immunoassay to detect the presence of an analyte in aliquid sample. Various ELISA formats are known in the art and any ofthese can be used in conjunction with the sulfonic acid stop reagent ofthe invention. Although antibodies are typically used in ELISAs, anysort of analyte-binding member can replace the antibody to provideanalyte-specific interaction. As such, kits, composition, and methods ofthe invention can be used with any enzyme-linked solid phase analytebinding assay. The methods of the invention can include anyanalyte-binding reagent immobilized on the solid phase, along with achromogenic reagent that can be detected using colorimetric analysis inthe presence of an enzyme. The invention is not limited to any type ofanalyte binding assay, or any type of analyte binding member, but someexamples are discussed to illustrate aspects of the invention. Theparticular immunoassay format employed will depend on the particularanalyte characteristics, the sample characteristics, the availablereagents, and the like.

One example of a common ELISA format is the “direct, antigen down” ELISAwhich is often used when the biological substance measured is antibody.In this format a purified protein (the antigen) is absorbed to theplastic surface of a multi-well plate. The plate is washed to removeexcess antigen and then blocked with a blocking agent that can beprotein based or synthetic, to prevent non-specific binding of theantibody. Next, a sample is added to the well that includes antibodiesthat specifically bind the antigen immobilized on the plastic surface.Excess sample is then washed off. After washing, secondary antibodyconjugated to a peroxidase enzyme specific for the animal antibody inthe biological sample is added. For example, if the sample is human, ananti-human antibody (e.g., an anti-human IgG) is used for detection. Achromogenic reaction can then be performed by adding a chromogenicsubstrate and a reactant, such as hydrogen peroxide, for the peroxidase.A sulfonic acid stop reagent of the invention can then be added to thechromogenic reaction, and colorimetric analysis can be performed using aspectrophotometer.

In other cases, for example, a “sandwich” ELISA can be performed byfirst immobilizing the analyte binding member (e.g., an analyte“capture” antibody) on the solid substrate, such as a plastic well. Theplastic surface is then typically blocked with a non-specific protein toprevent adherence of components from the biological sample when added tothe well in a next step. Analyte from the biological sample added to thewell specifically interacts with the immobilized antibody on the plate.The plate is washed and then a solution of a second analyte bindingmember (such as an antibody conjugated to a peroxidase enzyme, called adetection antibody) is added which interacts with the analyte alreadyimmobilized by the plastic bound antibody. The analyte thereforeeffectively becomes “sandwiched” between the antibody absorbed on theplate, and the enzyme-conjugated antibody. A chromogenic reaction canthen be performed by adding a chromogenic substrate and a reactant, suchas hydrogen peroxide, for the peroxidase. A sulfonic acid stop reagentof the invention can then be added to the chromogenic reaction, andcolorimetric analysis can be performed using a spectrophotometer.

Alternatively, the “sandwich” approach can be performed by first mixingan enzyme-conjugated antibody with a biological sample having ananalyte. The enzyme-conjugated antibody is used in excess andanalyte-enzyme-conjugated antibody complex forms in the sample. Thesample is then transferred to a well of an assay plate that has theanalyte capture antibody immobilized thereon. The analyte captureantibody binds the analyte-enzyme-conjugated antibody complex, and thenthe well can be washed to remove non-bound material. A chromogenicreaction can then be performed and then a sulfonic acid stop reagent canbe added, as described.

Kits of the invention can include one or more components, other than thesulfonic acid stop reagent and the chromogenic substrate, useful forperforming an ELISA. The kit can include optional components such asanalyte-binding members like antibodies, enzymes (e.g., HRP-linkedanti-IgG antibodies, etc), and enzyme substrates. The kit can alsoinclude vessels, such as multi-welled plates, in which the chromogenicreaction can take place and be analyzed. The kit can also include asolubility-enhancing component.

As described herein and in some aspects, the assay uses a compoundhaving specific affinity for the analyte. A compound with specificaffinity is referred to herein as a “binding moiety,” which refers toany sort of chemical group that can bind or interact with the analyte.The binding moiety can include naturally occurring molecules orderivatives of naturally occurring molecules, or synthetic molecules,such as small organic molecules, or larger synthetically preparedmolecules, such as polymers. Examples of binding moieties includepolypeptides, nucleic acids, polysaccharides, and portions of thesetypes of molecules that can bind a target species. Nucleic acids such asoligonucleotides that have a length sufficient to undergo complimentaryhybridization to a target nucleic acid analyte in a sample can be usedas the analyte binding moiety.

The binding moiety can be an antibody, which is a protein thatrecognizes a particular epitope on the analyte. In this regard, theanalyte can also be referred to as an “antigen” as it is common forantibody-antigen interactions to be described. An antibody can be apolyclonal antibody, a monoclonal antibody, or a genetically engineeredmolecule capable of binding the corresponding member of a specificbinding pair. One class of polypeptides that can be used as a bindingmoiety in the invention includes antibodies and antibody fragments.

Antibody and antibody fragments having specificity towards desiredanalytes are commercially available or can be prepared by techniquesknown in the art. For example, monoclonal antibodies (mAbs) can beobtained by any technique that provides for the production of antibodymolecules by continuous cell lines in culture. These include, forexample, the hybridoma technique (Kohler and Milstein, Nature,256:495-497 (1975)); the human B-cell hybridoma technique (Kosbor etal., Immunology Today, 4:72 (1983); and the EBV-hybridoma technique(Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96 (1985)). Such antibodies can be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof.

Fab or Fab′2 fragments can be generated from monoclonal antibodies bystandard techniques involving papain or pepsin digestion, respectively.Kits for the generation of Fab or Fab′2 fragments are commerciallyavailable from, for example, Pierce Chemical (Rockford, Ill.).

In some embodiments the analyte binding member comprises a nucleic acid.For example, a nucleic acid having a sequence capable of hybridizingwith a nucleic acid analyte under stringent conditions can beimmobilized on a plastic or glass surface and used in the kit or methodof the invention. Nucleic acid from a sample, such as a biologicalsample, can be placed in contact with the surface with immobilizednucleic acid. Nucleic acid analyte can be present from a cell samplethat is treated to disrupt the cells and optionally enrich the analytenucleic acid. Analyte nucleic acid can undergo hybridization bycomplimentary bonding to the immobilized nucleic acid. Stringenthybridization conditions are well known in the art (see for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).Presence of the analyte can then be detected with a free probe(non-immobilized) nucleic acid that can hybridize to the nucleic acidanalyte and that is either directly attached to an enzyme, or that canbe indirectly coupled to an enzyme.

In other modes of detection, a nucleic acid from a sample is immobilizedon a glass or plastic substrate, and then presence of the analyte can bedetected with a free probe (non-immobilized) nucleic acid that canhybridize to the nucleic acid analyte and that is either directlyattached to an enzyme, or that can be indirectly coupled to an enzyme.

In some cases, the binding moiety is conjugated to another compoundwhich facilitates the colorimetric analysis, such as described herein.

In some aspects of the invention, the methods, compositions, or kits ofthe invention include an oxidase or a peroxidase. As a general matter,an oxidase or a peroxidase can be used to generate a radical resultingin the transfer of electrons from one component to another. When thechromogenic substrate is present, it becomes oxidized to a chromogenicproduct which is used in conjunction with the sulfonic acid stop reagentof the invention.

In some cases the oxidase or peroxidase is coupled to a binding moietyfor the detection of an analyte. In these cases, the oxidase orperoxidase can simply serve to cause reduction of a peroxide compound,such as hydrogen peroxide, providing a chemical environment for theoxidation of the chromogenic substrate to a desired colored reactionproduct. Commercially available peroxidase conjugates, such as thosedescribed herein, can be used.

In other cases the oxidase or peroxidase has specificity towards ananalyte of interest in the biological sample. For example, glucoseoxidase (GOx) (EC 1.1.3.4) is an oxido-reductase that catalyses theoxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. Inorder to quantitate the amount of glucose in a biological sample,glucose oxidase and a peroxidase, such as horseradish peroxidase, can beadded directly to the sample. Glucose oxidase, which causes theformation of hydrogen peroxide, can be reduced to provide conditionsresulting in the oxidation of the chromogenic substrate. Xanthineoxidase is another enzyme that generates hydrogen peroxide by usinghypoxanthine and xanthine as substrates. L-gulonolactone oxidase (EC1.1.3.8) catalyzes the reaction of D-glucuronolactone toL-xylo-hex-3-gulonolactone and hydrogen peroxide. Using this type ofapproach, the analyte or the analyte binding member does not have beimmobilized on a solid surface, such as performed using ELISA. Otheroxidases include galactose oxidase, hexose oxidase, and pyranoseoxidase.

Peroxidase enzymes useful for the methods of the invention can beobtained from a variety of sources, such as plants. Common, commerciallyavailable peroxidases are from horseradish and soybean. Horseradishperoxidase has an approximate molecular weight of 44 kDa, and is asingle chain polypeptide glycoprotein with disulfide bridges thatincludes hemin plus Ca²⁺. HRP specific activity can be expressed inpyrogallol units (one pyrogallol unit will form 1.0 mg purpurogallinfrom pyrogallol in 20 sec at pH 6.0 at 20° C.) or ABTS units (one ABTSunit will oxidize 1 mmole of ABTS per minute at 25° C. at pH 5.0). HRPcan be inhibited by sodium azide, L-cystine, dichromate,ethylenethiourea, hydroxylamine, sulfide, p-aminobenzoic acid, Cd⁺²,Co⁺², Cu⁺², Fe⁺³, Mn⁺², Ni⁺², and Pb⁺².

Other peroxidases include lactoperoxidase, microperoxidase, NADHperoxidase NADPH peroxidase, fatty-acid peroxidase, and catalase.

In some modes of practice, a “peroxidase conjugate” is used in themethods for detecting an analyte. A conjugate generally refers to acompound that comprises two substances, wherein one of the substances iscoupled to the other. Coupling of the conjugate can be covalent ornon-covalent. A peroxidase conjugate can be one where a peroxidaseenzyme is coupled to a binding moiety that detects the analyte, or thatdetects the binding moiety that detects the analyte. Exemplaryperoxidase conjugates that can be commercially obtained includeavidin-peroxidase conjugates, monoclonal anti-FLAG™ M2-peroxidaseconjugates, anti-glutathione-S-transferase(GST)-peroxidase conjugates,anti-mouse IgG-peroxidase conjugates, anti-goat/sheep IgG-peroxidaseconjugates, Protein G-peroxidase conjugates, Protein-A peroxidaseconjugates, anti-rabbit IgG-peroxidase conjugates, andstrepavidin-peroxidase conjugates, from, for example, Sigma-Aldrich.

A biological sample that includes an analyte can be placed in ananalysis vessel, such as a well in a multiwall plate, so the analyte canbe immobilized and then detected using methods of the invention.Typically, steps for the immobilization of the analyte or analytebinding member (e.g., antibody), binding of analyte binding member tothe analyte, and subsequent binding of the analyte-analyte bindingmember complex can be performed using a suitable solution(s), includingincubation, blocking, and washing buffers. The solution can be anaqueous buffered solution, to maintain absorption of the analyte and/oranalyte binding member on the plastic surface, and maintain proteinconfiguration for proper binding and enzymatic activities. The blockingsolution can include a non-specific protein such as bovine serumalbumin, which can effectively block vessel binding sites that remainfollowing initial coating steps in ELISA procedures (e.g. 5% BSA-PBS).Washing buffers can include a surfactant such as Tween (an exemplarywashing buffer, PBS-T, contains 10 mM phosphate buffer pH 7.4, 150 mMNaCl, and 0.05% Tween 20).

In some modes of practice, an analysis vessel (such as a multi-wellplate) is provided that has an amount of peroxidase immobilized on asolid surface of the vessel. Again, the peroxidase can be immobilized byits conjugation to a binding member, with the binding member directly orindirectly bound to an analyte previously immobilized on the vesselsurface, using either an analyte binding member, or by the analyte beingabsorbed directly on the surface of the vessel. The amount ofimmobilized peroxidase can correlate with the amount of immobilizedanalyte, which correlates with the amount of analyte present in asample. In control wells, standards with known amounts of analyte can beused.

In order to explain modes of practicing of the invention, in exemplaryembodiments, analyte immobilization results in the correspondingimmobilization of picrogram quantities of a peroxidase enzyme, such ashorseradish peroxidase, on a surface of a single well of a 96-wellplate, such as an amount in the range of about 0.1 pg to about 100 ng(100000 pg), about 1 pg to about 10 ng (10000 pg), or about 1 pg toabout 1 ng (1000 pg). In this case, the peroxidase enzyme is immobilizedprior to adding the enzyme substrate and chromogenic reagent.

As a general matter, a peroxide substrate is added to the peroxidase,which is immobilized on a surface of the vessel, or present in solutionin the vessel. A cost-effective substrate for carrying out peroxidasereactions is hydrogen peroxide. HRP combines with hydrogen peroxide(H₂O₂) and can carry out heterolytic cleavage of the H₂O₂ oxygen-oxygenbond. The complex can oxidize a wide variety of chromogenic substrates,and then the reaction can be stopped by the addition of a sulfonic acidstop reagent such as one described herein.

Hydrogen peroxide and the chromogenic substrate can be present in areaction solution, which can be added to a reaction vessel that includesthe peroxidase enzyme. The reaction solution can be prepared fromcomponents of a kit that include the chromogenic substrate, hydrogenperoxide, reaction buffer. In some embodiments the hydrogen peroxide andchromogenic substrate are supplied in a ready-to-use one componentcomposition or solution. In other embodiments the chromogenic substrateand hydrogen peroxide are provided to a user separately, in a kit. Forexample, hydrogen peroxide can be provided as a concentrated stocksolution (e.g., ˜2-3%). The concentrated hydrogen peroxide can bediluted in the reaction solution to exemplary amounts in the range ofabout 0.1 mM to about 50 mM (˜3×10⁻⁴% to ˜0.17%), or about 0.5 mM toabout 10 mM (˜0.0017% to ˜0.034%). The kit can also include a reactionbuffer, such as sodium phosphate (pH 7-8) provided in dry orconcentrated form. For example, the buffer can be supplied as a 5× or10× concentrate which can then be diluted in the reaction composition toa working concentration, such as in the range of about 10 mM to about 75mM.

The chromogenic substrate can optionally be reconstituted from dry formor diluted from a stock solution and then added to the reactionsolution. Depending on the type of chromogenic substrate, the kit canoptionally include one or more different liquids, such asdimethylsulfoxide (DMSO), if dissolution of the chromogenic substratewould be facilitated using such components. The working concentration ofthe chromogenic substrate can be chosen based on factors such as thetype of chromogenic substrate, the amount of hydrogen peroxide, and/orthe amount of analyte (and corresponding peroxidase) in the sample beinganalyzed. Exemplary ranges of the chromogenic substrate are from about0.5 μM to about 10 mM, from about 10 μM to about 5 mM, or about 100 μMto about 3 mM.

Optionally, a cosolvent can be included in the reaction composition withthe analyte, enzyme, and chromogenic substrate. The cosolvent can ensurethe reagents of the reaction composition be maintained in soluble formand do not precipitate out of solution during the reaction, or later,such as upon addition of the stop reagent. Exemplary co-solvents includealcohols (i.e. methanol, ethanol, propanol), polyalcohols (i.e.glycerol, propylene glycol), dimethylsulfoxide, dimethylformamide,acetonitrile and similar solvents. Other solvents to improve solubilityare well known in formulation art for in vitro diagnostic applications,and are included herein. A cosolvent can be used in the reactioncomposition, at exemplary concentrations in the range of about 1% (v/v)to about 50% (v/v), or about 5% (v/v) to about 25% (v/v).

A solubility-enhancing components such as described in U.S. ProvisionalNo. 61/751,652 (Jan. 11, 2013; Lundquist) can optionally be included inthe reaction composition.

In some modes of practice, the reaction solution that includes thecomponents sufficient for color development of the chromogenic substratecan then be added directly to the vessel that includes the peroxidaseenzyme. Alternatively, components of the kit can be mixed directly inthe wells. Using multiwall plates, the reaction solution can bedispensed, for example, by pipetting, in the well manually or usingautomated apparatus. Multi-tip pipetting apparatus can be used toincrease the speed of the dispensing and or mixing process. Using a96-well plate format, typical reaction volumes range from about 25 μL toabout 200 μL, or from about 50 μL to about 150 μL.

The peroxidase reaction resulting in the conversion of the chromogenicsubstrate into the chromogenic product can be carried out for a desiredperiod of time at a desired temperature. The reaction can be monitoredvisually or spectrophotometrically to determine the development of colorin the composition. The incubation step will typically occur at roomtemperature, although a temperature in the range of about 10° C. toabout 50° C. can be employed. Incubation times will typically range fromabout 1 to about 60 minutes, or more usually about 5 to about 45minutes.

In order to describe aspects of the invention,3,3′,5,5′-tetramethylbenzidine (TMB) is used as the chromogenicsubstrate in the presence of an immobilized HRP enzyme (associated withthe analyte) and hydrogen peroxide. TMB, in the presence of HRP and H₂O₂is oxidized to form a blue product (λ_(max) 652), as previouslydescribed. The reaction can be observed visually, or the absorbance ofthe reaction can be monitored spectrophotometrically (e.g., at about 650nm) to obtain an absorbance value corresponding to the blue color. Thestop reagent can be added when the reaction reaches a desired color, orwhen a certain absorbance value is reached. For example, in the case ofTMB oxidation, or other chromogenic substrates that have similarabsorbance properties upon oxidation, the stop reagent can be added tothe reaction composition when the wells with the highest signals reachan absorbance of up to about 1.6 absorbance units at about 650 nm.Addition of the stop reagent converts the blue reaction product to ayellow product with an absorbance maxima at about 450 nm. The extinctioncoefficient of the yellow TMB product is about 2.3 times that of theblue TMB. Allowing the blue product to develop to about 1.5 or 1.6absorbance at 650 nm results in an absorbance between 3.5 and 4absorbance units upon the addition of stop reagent, which represents apractical high end for absorbance readings.

A sulfonic acid stop reagent of the invention, such as a compound ofFormula I (i.e., R—SO₃H, wherein R is defined herein) is then added tothe reaction composition. In exemplary modes of practice, the reagent ismethanesulfonic acid or sulfamic acid, or combinations thereof. The stopreagent is soluble in the reaction composition and mixing can thereforebe rapid. In an exemplary mode of practice, the stop reagent is 2×concentrated, or thereabouts, so the volume of stop reagent added to thewell is approximately the same as the reaction volume in the well. Forexample, 100 μL of stop reagent is added to 100 μL of the reactioncomposition. Exemplary concentrations of stop reagent are in the rangeof about 1.5× to about 5×, which can be used to facilitate rapid mixingof the stop reagent and reaction components.

In some aspects the stop reagent is added to the reaction composition toprovide a final concentration of stop reagent in the range of about 0.02M to about 2.0 M, about 0.02 M to about 1 M, about 0.02 M to about 0.5M, or about 0.02 to 0.2 M.

After the addition of the stop reagent, the reaction composition canoptionally be described in terms of its pH. The acidic nature of thestop reagent generally results in a lowering of the pH of the reactioncomposition after it is added. In some cases the pH of the reactioncomposition is in the range of about 0.2 to about 4, or morespecifically in the range of about 0.5 to about 2.

Optionally, depending on the particular stop reagent used, and also thetype of chromogenic substrate, a cosolvent can be included in the stopreagent composition. As previously discussed, the cosolvent can ensurethe reagents of the reaction composition be maintained in soluble formand do not precipitate out of solution upon addition of the stopreagent. Co-solvents such as alcohols, polyalcohols, dimethylsulfoxide,dimethylformamide, acetonitrile and similar solvents as previouslydescribed, can be used at a final concentration following addition ofthe stop reagent in the range of about 1% (v/v) to about 50% (v/v), orabout 5% (v/v) to about 25% (v/v). In methods using TMB as thechromogenic compound, the color of the reaction composition cantransition to yellow corresponding to the two-electron oxidation productof TMB (λ_(max) 450). The stop reagent of the invention can stabilizethe two-electron oxidation product, resulting in a yellow compositionthat maintains high levels of absorbance at about 450 nm when monitoredspectrophotometrically.

Analysis of the reaction mixture is performed under “stopped”conditions. By this it is meant that the reaction is allowed to proceedfor a predetermined period and then terminated with a stop reagent ofthe invention. Without being bound by theory, it is thought that in theleast, the addition of the stop reagent terminates the reaction by beinga non-specific inhibitor of the enzyme, such as to sequester metal ionsthat are normally present for enzyme activity. However, the sulfonicacid stop reagent of the invention offers an advantage in that itstabilizes the chromogenic reaction product after the reaction has beenstopped. For example, the stop reagents of the invention can stabilizethe color of the chromogenic reaction product so that it minimizeschange in absorbance characteristics of the chromogenic reaction productafter it is added.

For example, with reference to FIG. 1, in the presence of stop reagentsof the invention such as 0.2 M sulfamic acid or 0.15 M methanesulfonicacid, the stopped compositions exhibit nominal loss of absorbance at 450nm, as compared to conventional stop reagents of 0.5 M H₂SO₄, 1 Mphosphoric acid, or 0.25 M HCl, over extended periods of time. This canprovide substantial improvements in the quality of analyte analysis, andlead to more accurate measurements of the amount of analyte in a sample.When analysis is performed using a multi-well format (such as a 96-wellplate) the amount of time needed to add and mix the stop reagent intothe wells may be significant, especially if analysis is performed usingmanual pipetting procedures. The loss of absorbance seen usingconventional approaches (e.g., using 0.5 M H₂SO₄) can lead tosample-to-sample variations that may not accurately represent the actualamount of analyte in a sample. Use of the stop reagents of the inventionsignificantly minimizes the loss of absorbance, resulting in moreaccurate measurements of analyte amounts. This also affords the usermore time to carry out the assays, which can allow the throughput of theassays (i.e., the number of assays performed) within a single reactionbatch to be increased.

Optionally, aspects of the invention can be described in the capacity ofthe stop reagent to stabilize the chromogenic product over a determinedperiod of time with reference to absorption properties of thechromogenic product. For example, in some aspects the stop reagentstabilizes the chromogenic substrate so that it exhibits less than a 15%reduction, less than a 10% reduction, or less than a 5% reduction inabsorbance at 60 minutes after addition of the stop reagent; less than a20% reduction, less than a 15% reduction, or less than a 10% reductionin absorbance at 120 minutes after addition of the stop reagent; or lessthan a 30% reduction, less than a 25% reduction, or less than a 20%reduction in absorbance at 180 minutes after addition of the stopreagent

In some methods of analyzing, such as using a direct or sandwichimmunoassay as described herein, and the reagents employed, theabsorbance of the chromogenic product as determined by aspectrophotometric method will be directly proportional to the amount ofanalyte in the sample. Where one is interested in a qualitative resultor a semi-quantitative result, such as determining whether the amount ofanalyte is above a predetermined threshold, versus determining theconcentration of analyte, the amount of peroxidase enzyme and/orchromogenic substrate can be selected to provide a clear signal ascompared to the absence of analyte or analyte below the predeterminedvalue.

For quantitation, absorbance can be accurately measured usingappropriate hardware and software if desired. Controls can be employed,where the signal to concentration of the analyte is determined, so thatthe signal can be directly related to the concentration of analyte inthe assayed sample. In this manner, both the presence and the amount ofanalyte in the sample can be determined. Simple spectrophotometers, suchas UV/VIS spectrophotometers for wavelengths between 175 nm and 900 nmcapable of determining the absorbance of a sample are commerciallyavailable, for example, from Perkin Elmer. In analyzing the sample, alight of a specific wavelength, such as selected by an optical filter ormonochromator, is transmitted through the sample, and a detectormeasures the percentage of the initial transmitted through the sample.The amount of transmitted light is generally inversely proportional tothe amount of analyte in the sample.

In other modes of practice, analysis is performed using a microplatereader. A variety of microplate readers capable of accommodating andanalyzing the absorbance of samples in the wells of 96-well plates, arecommercially available, from, for example BioTek (Winooski, Vt.).

Example 1

Methanesulfonic acid and sulfamic acid were compared to conventionalstop reagent acids of sulfuric acid, hydrochloric acid and phosphoricacid to stop and stabilize a HRP TMB reaction.

To aliquots of 12 mL of TMB substrate (TMBW, SurModics, Inc.) were addedeither 35 μL or 5 μL of horseradish peroxidase (HRP) at 1 μg/ml. Thesolutions were mixed and the substrate plus HRP was distributed into a96 well plate (100 μL/well). This resulted in TMB containing two HRPlevels, 300 pg/well or 40 pg/well. One hundred microliters of TMBW wasalso added to a set of wells as no HRP blanks. After the blue color haddeveloped so that the 300 pg/well conditions had an absorbance ofapproximately 1.5 at 650 nm (approximately 15 minutes), 100 μL of thestop reagents were added. The absorbance at 450 nm was them monitoredfor 3 hours with a reading every minute. There was an N of three foreach HRP level/stop reagent combination and an N of 2 for the blanks.

Table 2 and FIGS. 1 and 2 summarize the experimental results. Thesulfonic acids had much more stabile signals for the entire 180 minutes.Table 1 provides ratios of the signal after three hours compared to thesignal at 5 minutes after addition of the stop reagent. Themethanesulfonic acid and sulfamic acid showed more stabile signals thanthe reference acids, and the differences were more pronounced at thehigher HRP level.

TABLE 2 Values are absorbance (A450) at High HRP Low HRP no HRP 180minutes divided by 5 minutes (300 pg/well) (40 pg/well) (Blank) 0.5MH2SO4 (Reference) 0.41 0.53 1.00 1M phosphoric (Reference) 0.79 0.701.07 0.25M HCl (Reference) 0.57 0.63 1.04 0.2M sulfamic acid (Invention)0.90 0.76 1.06 0.15M methanesulfonic acid 0.90 0.76 1.11 (Invention)

Example 2

A model assay that used biotinylated mouse IgG as the analyte wasperformed to investigate the stability of the resulting standard curveswith the different stop reagents. Antibodies and streptavidin-HRP wereobtained from Jackson Immunoresearch. Plates were coated with rabbitanti-mouse antibody (0.1 μg/well). Coated plates were incubated with adilution series of biotinylated mouse IgG for 2 hours. The plates werethen washed with PBS-Tween and then incubated for 20 minutes withstrepavidin-HRP (1 μg/mL). After washing, 100 μL of TMB substrate wasadded to each well. The plate was allowed to develop until the highestabsorbances were approximately 1.5 at 650 nm (approximately 15 minutes)and then 100 μL of the different stop reagents were added.

Three stop reagents were used: 0.15 M methanesulfonic acid, 0.2 Msulfamic acid and 0.5 M sulfuric acid. The absorbance at 450 nm was thenmonitored for eight hours, reading every 2.5 minutes. There was an N offour for each mouse IgG level/stop reagent combination. FIGS. 3-5 showthe mouse IgG standard curves (4PL Fits) generated at selected timepoints. FIG. 3 is the result for the sulfuric acid comparison whichshows that even after 30 minutes the ability to accurately quantitateacross the across the dynamic range cannot be performed at higheranalyte concentrations. FIGS. 4 and 5 demonstrate the results formethanesulfonic acid and sulfamic acid. The standard curves are verystabile for two hours and are also well fitted even eight hours afterthe addition of the stop reagent.

Example 3

A HRP assay was performed comparing other sulfonic acids to sulfuricacid in an HRP assay. Experiments were done using HRP alone as describedin Example 1, except that the plates were read for only 90 minutes.Tables 2-4 report the results for the 300 pg/well condition where thestability difference is most pronounced.

As reflected in Table 3, para-toluenesulfonic acid (p-TSA) and1-acrylamido-2 methylpropanesulfonic acid (AMPS) were used as stopreagents and compared to sulfuric acid. The p-TSA caused precipitationof reagent in the wells when it was dissolved in water alone. TMB wasable to be maintained in solution by the addition of 20% DMSO to thestop reagent solution without adverse affects on the reaction or abilityto analyze the reaction product. Tables 4 and 5 demonstrate that bothethanesulfonic acid and propanesulfonic acid also generate more stabilesignals than the sulfuric acid reference conditions.

TABLE 3 A450 at 5 A450 at 90 90 minute/5 Stop Reagent pH minutes minutesminute ratio 0.5N H2SO4 (reference) 3.398 2.548 0.750 0.2Mp-toluenesulfonic acid 0.93 3.441 2.652 precipitated 0.2Mp-toluenesulfonic 1.02 2.903 2.894 0.997 acid in 20% DMSO/water 0.2M1-acrylamido-2 1.04 3.306 3.105 0.939 methylpropanesulfonic acid (AMPS)

TABLE 4 A450 at 5 A450 at 90 90 minute/5 Stop Reagent pH minutes minutesminute ratio 0.5N H2SO4 (reference) 3.540 2.241 0.633 0.1Methanesulfonic acid 1.16 3.392 3.194 0.942 0.15M ethanesulfonic acid1.01 3.445 3.251 0.944 0.2M ethanesulfonic acid 0.9 3.452 3.262 0.9450.15M methanesulfonic acid 0.99 3.486 3.292 0.945

TABLE 5 A450 at 5 A450 at 90 90 minute/5 Stop Reagent pH minutes minutesminute ratio 0.5N H2SO4 (reference) 3.466 1.679 0.485 0.1Mpropanesulfonic acid 1.16 3.355 3.149 0.939 0.15M propesulfonic acid1.01 3.411 3.215 0.943 0.2M propanesulfonic acid 0.9 3.427 3.234 0.9440.15M methanesulfonic acid 0.99 3.408 3.226 0.947

1. A composition for colorimetric analysis comprising an enzyme; achromogenic reaction product; and a sulfonic acid that is a compound ofFormula I:R—SO₃H wherein R is methyl, ethyl, propyl, —NH₂, or mixtures thereof. 2.The composition of claim 1 wherein the sulfonic acid is present in anamount in the range of 0.02 M to 2.0 M.
 3. The composition of claim 2wherein the sulfonic acid is present in an amount in the range of 0.02to 0.5 M
 4. The composition of claim 1 wherein the pH of the compositionis in the range of 0.2 to
 4. 5. The composition of claim 1 wherein thechromogenic reaction product is an oxidized benzidine compound offormula III:

wherein X, X′, Y, Y′, R, and R′ are independently hydrogen, alkyl,alkoxy, or combinations thereof, wherein the alkyl or alkoxy group(s),if present, have four or less carbon atoms.
 6. The composition of claim1 wherein the enzyme is a peroxidase or an oxidase.
 7. The compositionof claim 6 wherein the peroxidase or oxidase is selected from the groupconsisting of horseradish peroxidase, soybean peroxidase, glucoseoxidase, galactose oxidase, xanthine oxidase, lactoperoxidase,microperoxidase, NADH peroxidase NADPH peroxidase, fatty-acidperoxidase, and catalase.
 8. The composition of claim 1 comprisinghorseradish peroxidase; oxidized tetramethylbenzidine; and sulfamicacid, methanesulfonic acid, or ethanesulfonic acid.
 9. A method ofconducting colorimetric analysis comprising steps of: (a) providing areaction composition comprising an enzyme and a chromogenic substrate;(b) allowing the reaction composition to form a chromogenic reactionproduct; (c) adding a sulfonic acid to the composition comprising thechromogenic reaction product, wherein the sulfonic acid is a compound ofFormula I:R—SO₃H wherein R is methyl, ethyl, propyl, —NH₂, or mixtures thereof;and (d) after step (c), performing colorimetric analysis on thecomposition comprising the reaction product.
 10. The method of claim 9wherein chromogenic substrate is present in the composition in the rangeof 0.5 μM to 10 mM.
 11. The method of claim 9 wherein step (d) comprisesreading a wavelength absorption of the composition, wherein thewavelength absorption is about 450 nm.
 12. The method of claim 9 whereinthe method is an enzyme-linked immunosorbent assay (ELISA) and in step(a) the composition further comprises an antibody and an analyte. 13.The method of claim 9 where, in step (c), the sulfonic acid is added tothe composition in an amount in the range of 0.02 M to 2.0 M.
 14. Themethod of claim 9 wherein the composition formed in step (c) is thecomposition of claim
 8. 15. A kit for colorimetric analysis comprising;a chromogenic substrate capable of forming a chromogenic reactionproduct, and a sulfonic acid that is a compound of Formula I:R—SO₃H wherein R is methyl, ethyl, propyl, —NH₂, or mixtures thereof.16. The kit of claim 15 further comprising one or more of components(a), (b), (c), or (d): (a) a peroxidase, an oxidase, or a conjugate of aperoxidase or an oxidase; (b) an analyte specific binding member; (c) ananalyte positive control, an analyte negative control, or mixturesthereof; (d) an analysis plate upon which colorimetric analysis can beperformed.
 17. The kit of claim 15 including instructions to add thesulfonic acid to a composition comprising an enzyme and chromogenicreaction product
 18. The kit of claim 15 wherein the sulfonic acid, thechromogenic substrate, or both, are provided in dry form or are providedin a solvent.
 19. The kit of claim 15 wherein the chromogenic substrateis a benzidine compound of formula III:

wherein X, X′, Y, Y′, R, and R′ are independently hydrogen, alkyl,alkoxy, or combinations thereof, wherein the alkyl or alkoxy group(s),if present, have four or less carbon atoms.
 20. A method for stopping areaction in a composition comprising a chromogenic reaction product, themethod comprising a step of: adding a sulfonic acid to a reactioncomposition comprising an enzyme and a chromogenic reaction product,wherein the sulfonic acid is a compound of Formula I:R—SO₃H wherein R is methyl, ethyl, propyl, —NH₂, or mixtures thereof,wherein the sulfonic acid stops reaction in the composition.